Targeting mutant p53-R248W reactivates WT p53 function and alters the onco-metabolic profile

Abstract

TP53 is the most commonly mutated gene in cancer, and gain-of-function mutations have wide-ranging effects. Efforts to reactivate wild-type p53 function and inhibit mutant functions have been complicated by the variety of TP53 mutations. Identified from a screen, the NSC59984 compound has been shown to restore activity to mutant p53 in colorectal cancer cells. Here, we investigated its effects on esophageal adenocarcinoma cells with specific p53 hot-spot mutations. NSC59984 treatment of cells reactivated p53 transcriptional regulation, inducing mitochondrial intrinsic apoptosis. Analysis of its effects on cellular metabolism demonstrated increased utilization of the pentose phosphate pathway and inhibition of glycolysis at the fructose-1,6-bisphosphate to fructose 6-phosphate junction. Furthermore, treatment of cells with NSC59984 increased reactive oxygen species production and decreased glutathione levels; these effects were enhanced by the addition of buthionine sulfoximine and inhibited by N-acetyl cysteine. We found that the effects of NSC59984 were substantially greater in cells harboring the p53 R248W mutation. Overall, these findings demonstrate p53-dependent effects of NSC59984 on cellular metabolism, with increased activity in cells harboring the p53 R248W mutation. This research highlights the importance of defining the mutational status of a particular cancer to create a patient-centric strategy for the treatment of p53-driven cancers.

Keywords: NSC59984; gain-of-function; metabolism; mutant p53; p53-R248W.

Figure 1

NSC59984 treatment re-activates WT-p53 signaling and intrinsic apoptosis. (A) Panel of EAC cells studied, providing their biological origin and associated TP53 status. (B) Cellular proliferation was determined by fold change in CyQUANT measurement of cellular DNA following treatment with NSC59984 (12 μM) for 72 hr over carrier control in EAC cells. Fold change in carrier-treated cells was normalized to 1. A 2-way ANOVA test with Tukey correction was carried out for statistical analysis. (C) Analysis of apoptosis in EAC cells as measured by change in percentage of Annexin V positive cells following treatment with NSC59984 (12 μM) for 72 hr. A 2-way ANOVA test with Tukey correction was carried out for statistical analysis. (D) Whole cell lysate from EAC cells was blotted for p53 and p73 expression following 8 hr treatment with NSC59984 (12 μM). β-Actin served as the loading control. Numbers represent fold change in expression relative to changes in β-Actin. (E) Fold change in mRNA expression of PUMA analyzed by RT-qPCR following 3 hr treatment NSC59984 (6, 12 or 25 μM). Expression was normalized to β-actin, and changes in carrier-treated cells were set to 1. A 2-way ANOVA test with Dunnett’s correction was carried out for statistical analysis. (F) Whole cell lysate from EAC cells was blotted for Bax and p21 expression following 8 hr treatment with NSC59984 (6, 12 or 25 μM). β-Actin served as the loading control. (G) ChIP analysis of the occupancy of p53 on the p21 promoter in carrier-treated cells or following 2 hr treatment with 12 μM NSC59984 in CP-A-WT or ESO26-R248W cells. ChIP was performed using the Active Motif ChIP-IT Express^® kit. Chromatin Input was diluted 1:100 before analysis (left), ChIP DNA was analyzed as percent of Input (right). Raw Ct values can be found in Supplemental Table 1. A 2-way ANOVA with Šidák’s correction was carried out for statistical analysis. * = 0.05, ** = 0.005, *** = 0.0005, **** = 0.00005.

Figure 2

GOF mutant p53 alters cellular metabolic profiles. (A) Basal mitochondrial respiration, as measured with the Seahorse XF^e96 Cell Mito Stress Test Kit, in the panel of EAC cells. Arrows indicate addition of metabolism-modulating drugs. (B) Basal glycolytic function, as measured with the Seahorse XF^e96 Glycolytic Stress Test Kit, in the panel of EAC cells. Arrows indicate addition of metabolism-modulating drugs. (C) Mitochondrial function was measured using the Seahorse XF^e96 Cell Mito Stress Test Kit following treatment with NSC59984 (12 μM) for 72 hr in EAC cells. (D) Glycolytic function was measured using the Seahorse XF^e96 Glycolytic Stress Test Kit following treatment with NSC59984 (12 μM) for 72 hr in EAC cells. * = 0.05, ** = 0.005, *** = 0.0005, **** = 0.00005.

Figure 3

Isogenic cells match the parental cell lines. (A) Basal mitochondrial respiration, as measured with the Seahorse XF^e96 Cell Mito Stress Test Kit, in p53-expressing KO cells. Arrows indicate addition of metabolism-modulating drugs. (B) Basal glycolytic function, as measured with the Seahorse XF^e96 Glycolytic Stress Test Kit, in p53-expressing KO cells. Arrows indicate addition of metabolism-modulating drugs. (C) Mitochondrial function was measured using the Seahorse XF^e96 Cell Mito Stress Test Kit following treatment with NSC59984 (12 μM) for 72 hr in p53-expressing KO cells. (D) Glycolytic function was measured using the Seahorse XF^e96 Glycolytic Stress Test Kit following treatment with NSC59984 (12 μM) for 72 hr in p53-expressing KO cells. * = 0.05, ** = 0.005, *** = 0.0005, **** = 0.00005.

Figure 4

Effects of NSC59984 treatment on specific aspects of mitochondrial function. (A) Analysis of mitochondrial membrane potential (ΔΨm) as measured by the ratio of JC-1 fluorescence following treatment with NSC59984 (12 μM) for 72 hr over carrier control in EAC cells. Fold change in carrier-treated cells was normalized to 1. (B) Fold-change in lactate secretion (mM per µg of cellular protein) following treatment with NSC59984 (12 μM) for 72 hr over carrier control in EAC cells. Fold change in carrier-treated cells was normalized to 1. C. Fold-change in total ROS levels (DCFDA) following treatment with NSC59984 (12 μM) for 72 hr over carrier control in EAC cells. Fold change in carrier-treated cells was normalized to 1. (D) Fold-change in total GSH level (µM GSH per µg of cellular protein) following treatment with NSC59984 (12 μM) for 72 hr over carrier control in EAC cells. Fold change in carrier-treated cells was normalized to 1. (E) Fold-change in glucose uptake following treatment with NSC59984 (12 μM) for 72 hr in EAC cells. (F) Fold-change in glucose utilization following treatment with NSC59984 (12 μM) for 72 hr over carrier control in EAC cells. Fold change in carrier-treated cells was normalized to 1. YSI analyzer measurements were normalized to CyQUANT measurement of cellular DNA. (A-F). A 2-way ANOVA test with Tukey correction was carried out for statistical analysis for each of these assays. * = 0.05, ** = 0.005, *** = 0.0005, **** = 0.00005.

Figure 5

Modulation of NSC59984 effects by combination treatment with ROS regulators. (A) Fold change in total ROS levels (DCFDA) following treatment of ESO26-R248W cells with NSC59984 (12 μM) for 24 hr either alone or in combination with NAC (1 mM) or BSO (10 mM) over carrier control. Fold change in carrier-treated cells was normalized to 1 (B) Fold change in total GSH level (µM GSH per µg of cellular protein) in ESO26-R248W cells treated with NSC59984 (12 μM) for 24 hr either alone or in combination with NAC (1 mM) or BSO (10 mM) over carrier control. Fold change in carrier-treated cells was normalized to 1. (C). ESO26-R248W cellular proliferation was measured following treatment with NSC59984 (12 μM) for 24 hr either alone or in combination with NAC (1 mM) or BSO (10 mM). Cells were re-seeded and fold change in CyQUANT quantitation of cellular DNA was measured following 5 days of growth over carrier control. Fold change in carrier-treated cells was normalized to 1. (D) Apoptosis in ESO26-R248W cells treated with NSC59984 (12 μM) for 24 hr either alone or in combination with NAC (1 mM) or BSO (10 mM) as measured by change in percentage of Annexin V positive cells. Cells were re-seeded and the change in percent of Annexin V positive cells was assessed following 72 hr growth. (E) Effect of NSC59984 treatment on G6PD protein level. Shown is a representative image, whole cell lysate was blotted for G6PD expression following 8 hr treatment with NSC59984 (12 μM) in EAC cells. (F) Fold change in G6PD activity following treatment with NSC59984 (12 μM) for 72 hr over carrier control in EAC cells. Fold change in carrier-treated cells was normalized to 1. (G) Fold-change in total NADPH levels following treatment with NSC59984 (12 μM) for 72 hr over carrier control in EAC cells. (H) Western blot analysis of p53-p73 protein levels. ESO26 cells were treated with NSC59984 (12 μM) for 72 hr before cell lysis. Total protein was IP with p53 D01. Sample Input, IgG preclear, unbound protein controls and IP samples were blotted for p73. Fold change in carrier-treated cells was normalized to 1. (A-H). A 2-way ANOVA test with Tukey correction was carried out for statistical analysis for each of these assays. (I) ChIP analysis of the occupancy of p73 on the CaN19 promoter in carrier-treated cells or following 2 hr treatment with 12 μM NSC59984 in CP-A-WT or ESO26-R248W cells. ChIP was performed using the Active Motif ChIP-IT Express^® kit. Chromatin Input was diluted 1:100 before analysis (left), ChIP DNA was analyzed as percent of Input (right). A 2-way ANOVA with Šidák’s correction was carried out for statistical analysis. Raw Ct values can be found in Supplemental Table 2 . * = 0.05, ** = 0.005, *** = 0.0005, **** = 0.00005.

Figure 6

NSC59984 treatment results in activation of the PPP. (A) Incorporation of [¹³C₆] glucose into cellular metabolites of ESO26-R248W cells following treatment with NSC59984 (12 μM) for 72 hr was assessed by ¹H-¹³C HSQC NMR. Spectra were normalized to total cellular protein content. B-D: Fold change in total cellular quantities of metabolite intermediates were assessed by IC-FTMS over carrier control set to 1 in: (B) Glycolysis pathway; (C) Pentose phosphate pathway; (D) Adenylates ATP, AMP, and ADP. (E) Fold change in Hexokinase activity following treatment with NSC59984 (12 μM) for 72 hr over carrier control in ESO26-R248W cells. Fold change in carrier-treated cells was normalized to 1. A-E. Paired t-tests were carried out for statistical analysis for each of these assays. * = 0.05, ** = 0.005, *** = 0.0005, **** = 0.00005.

Figure 7

NSC59984 treatment results in p53-dependent activation of TIGAR. (A) Fold change in PFK1 activity following treatment with NSC59984 (12 μM) for 72 hr over carrier control in ESO26-R248W cells. Fold change in carrier-treated cells was normalized to 1. A paired t-test was carried out for statistical analysis. (B) Fold changes in mRNA expression levels of TIGAR were analyzed by RT-qPCR following treatment with NSC59984 (12 μM) for 72 hr over carrier control in EAC cells. Fold change in carrier-treated cells was normalized to 1. Expression was normalized to β-actin. A 2-way ANOVA test with Tukey correction was carried out for statistical analysis. (C) Whole cell lysate was blotted for TIGAR expression following treatment with NSC59984 (12 μM) for 72 hr in the selected panel of EAC cells. (D) ChIP analysis of the occupancy of p53 on the TIGAR promoter in carrier-treated cells or following 2 hr treatment with 12 μM NSC59984 in CP-A-WT or ESO26-R248W cells. ChIP was performed using the Active Motif ChIP-IT Express^® kit. Chromatin Input was diluted 1:100 before analysis (left), ChIP DNA was analyzed as percent of Input (right). A 2-way ANOVA with Šidák’s correction was carried out for statistical analysis. Raw Ct values can be found in Supplemental Table 3 (E) Cellular proliferation in ESO26-R248W cells following treatment with NSC59984 (12 μM) for 72 hr over carrier control in siTIGAR or siControl cells. Proliferation was determined by change in CyQUANT measurement of cellular DNA. Fold change in carrier-treated cells was normalized to 1. A 2-way ANOVA test with Tukey correction was carried out for statistical analysis. (F) Western blot analysis of G6PD protein levels following treatment with NSC59984 (12 μM) for 72 hr in siTIGAR or siControl ESO26-R248W cells. * = 0.05, ** = 0.005, *** = 0.0005, **** = 0.00005.

Figure 8

Combination of NSC59984 with metabolic inhibitors shows improved effects. (A) Analysis of cellular proliferation over a period of 5 days following 48 hr treatment with either Metformin (5 mM), NSC59984 (12 μM) or a combination in EAC cells (B) Analysis of cellular proliferation over a period of 5 days following 48 hr treatment with either 2DG (5mM), NSC59984 (12 μM) or a combination in EAC cells. In all panels, cellular proliferation was measured by CyQUANT analysis of cellular DNA. A 2-way ANOVA test with Tukey correction was carried out for statistical analysis. * = 0.05, ** = 0.005, *** = 0.0005, **** = 0.00005.

Figure 9

Schematic detailing the effects of p53 stabilization on metabolic pathways. The stabilization of the p53 structure upon treatment with NSC59984 initiates a cascade of effects through both transcriptional- and non-transcriptional regulation. Via transcriptional regulation, stabilized p53 increases expression of TIGAR which causes a subsequent reduction of PFK and blocks glycolysis. A concurrent increase in G6PD potentially mediated by p73 having been released from an inhibitory complex with mutant p53 (dashed line) allows the processing of glucose to flow through the PPP via increased HK and Glucose-6P. This reduces cellular energetics and subsequently cellular proliferation. Simultaneously, a transcriptional increase in Bax allows increased cytochrome C release from the mitochondria with ensuing intrinsic apoptosis. Labels in BOLD indicate measured increases following treatment with NSC59984. * = 0.05, ** = 0.005, *** = 0.0005, **** = 0.00005.